Previously, we had discovered N-RAP, a novel, muscle specific protein with homology to nebulin. The C-terminal half of N-RAP contains more than two complete actin-binding super repeats similar to those found in nebulin. The N-terminus contains the consensus sequence of a cysteine rich LIM domain, which binds talin in vitro. Our work had localized N-RAP at the myotendon junction in skeletal muscle and at the intercalated disk in cardiac muscle, leading us to hypothesize that the N-RAP domains may be involved in linking the ends of myofibrils to specialized protein complexes found beneath the sarcolemma. We have been using ultrastructural localization and biochemical copurification to elucidate the organization and function of N-RAP. N-RAP is located at the cardiac intercalated disk in the vicinity of the terminal actin filaments of cardiac myofibrils, and remains tightly bound at the ends of isolated myofibrils after detergent extraction. N-RAP is localized in fragments of partially purified cardiac intercalated disks, which are also enriched for many junctional proteins. Detergent extraction and further purification yielded a fraction morphologically similar to fasciae adherentes that was highly enriched in N-RAP, N-cadherin, connexin-43, talin, desmin, and alpha-actinin. The finding that N-RAP copurifies with detergent-extracted intercalated disk fragments even though beta-integrin and vinculin have been completely removed suggests that N-RAP association with the adherens junction region is mediated by the cadherin system. Consistent with this, we found that defined regions of N-RAP can directly bind alpha-actinin in a blot overlay assay. We have also been assessing N-RAP's possible involvement in myofibril assembly using primary cultures of embryonic chick cardiomyocytes as a model system. Targeting and functional effects of N-RAP domains were studied by expression as GFP-tagged fusion proteins. GFP-tagged N-RAP was targeted to myofibril precursors, myofibril ends, and cell contacts, expression patterns similar to endogenous N-RAP. The GFP-tagged N-RAP LIM domain (GFP-N-RAP-LIM) was targeted to the membrane in cells with myofibril precursors and cell-cell contacts. The GFP-tagged super repeats (N-RAP-SR) and the GFP-tagged domain normally found in between the super repeats and the LIM domain (N-RAP-IB) were each observed at sites of myofibril assembly, incorporating into myofibril precursors in a manner similar to full length N-RAP. However, unlike full length N-RAP, N-RAP-SR and N-RAP-IB were also found in mature myofibrils, associating with the sarcomeric actin filaments and the Z-lines, respectively. N-RAP-IB was also colocalized with alpha-actinin at cell contacts. Each of the N-RAP constructs could inhibit the formation of mature myofibrils in cultured cardiomyocytes, with the effects of N-RAP-SR and N-RAP-IB depending on the time of transfection. The results show that each region of N-RAP is critical for myofibril assembly. Combining the targeting and functional effects of N-RAP domains with information in the literature, we proposed a new model for initiation of myofibrillogenesis in which N-RAP functions as an organizing center for key components. Finally, we have used yeast two-hybrid technology to identify proteins that may be potential partners in N-RAP function.